Seven New Phenylhexanoids with Antioxidant Activity from Saxifraga umbellulata var. pectinata

Seven new phenylhexanoids, (S)-(+)-3,4-dihydroxy-11-methoxyphenylhex-9-one (1), (E) 3,4-dihydroxy-phenylhex-10-en-9-one (2), (E)-4-hydroxyphenylhex-10-en-9-one (3), (R)-(−)-3,4,11-trihydroxyphenylhex-9-one 11-O-β-d-glucopyranoside (4), (R)-(−)-4,11-dihydroxyphenylhex-9-one 11-O-β-d-glucopyranoside (5), phenylhex-4,9,11-triol 11-O-β-d-glucopyranoside (6), and 9-O-acetyl-phenylhex-4,9,11-triol 11-O-β-d-glucopyranoside (7), were isolated and identified from Tibetan medicine Saxifraga umbellulata var. pectinate. The antioxidant activities of these compounds were evaluated using the DPPH and ABTS radical scavenging experiments. In the ABTS experiment, compounds 1 (IC50 13.99 ± 2.53 μM) and 2 (IC50 13.11 ± 0.94 μM) exhibited significantly better antioxidant activity than L-ascorbic acid (IC50 23.51 ± 0.44 μM).


Introduction
Saxifraga umbellulata var. pectinata, which belongs to the family Saxifragaceae, is a perennial herb mainly distributed on plateaus above 3000 m above sea level [1]. It is one of the major varieties of the traditional Tibetan medicinal herb called 'Songdi' [2]. Tibetan clinical medicine analyzes 'Songdi' in terms of the four qi and the five tastes. It is believed that 'Songdi' is cold, and the taste is bitter, and it is mostly used for the treatment of hepatobiliary (Tripa disease) and digestive diseases (Tripa enteropathy) [3]. 'Songdi' has a long history of medicinal use in Tibetan medicine, and is mostly used as the ruling medicine or in combination with other Tibetan medicines in Tibetan medical prescriptions. For example, the classical Tibetan prescription for hepatitis and cholecystitis, Ershiwuwei Songshi pills, uses 'Songdi' as one of the prescribed drugs [3].
To investigate potential active ingredients for the treatment of hepatitis, cholecystitis, and digestive system diseases, additional studies of the phytochemistry and biology of S. umbellulata var. pectinate were conducted.
As a result, seven novel phenylhexanoids ( Figure 1) were isolated and identified from S. umbellulata var. pectinata. This paper reports on the isolation, structural identification, and antioxidant effect of these compounds.

Identification of Compounds 1-7
1 was obtained as a yellowish oil, and its molecular formula was analyzed as C13H18O4 by HR ESI MS (m/z 237.1132 [M-H] − , calculated 237.1127 for C13H17O4). This indicates five degrees of unsaturation.
The above 1 H and 13 C NMR data (Table 1) of 1 indicate that there was a phenylhexyl skeleton in 1, the same as in inonophenol A [12]. The molecular formula of 1 was C13H18O4 (m/z 238), having one more -CH2-group (m/z 14) than inonophenol A (C12H16O4, m/z 224) Comparing the 1 H and 13 C NMR data of 1 with the data for inonophenol A, most of the data are similar, except for the C10, C11, C12, and -OCH3 data of 1.
The C11 signal (δC 74.6) in 1 was down-shifted by 9.5 chemical shift units compared to that in inonophenol A. Meanwhile, the C10 and C12 signals (δC 50.6 and 19.4) in 1 were up-shifted by 2.1 and 3.8 chemical shift units, respectively, compared to those in inonophenol A. These pieces of evidence prove that 1 resulted from the substitution of the hydroxyl group in inonophenol A with a methoxy group. The long-range correlations ( Figure 2) of δH 3.27 (3H, s, -OCH3) with δC 74.6 (C11) and δH 3.78 (1H, dqd, J =7.5, 6.2, 5.2 Hz, 11-H) with δC 56.8 (-OCH3) in the HMBC spectrum of 1 further support this inference 1 13   [12]. The molecular formula of 1 was C 13 H 18 O 4 (m/z 238), having one more -CH 2 -group (m/z 14) than inonophenol A (C 12 H 16 O 4 , m/z 224). Comparing the 1 H and 13 C NMR data of 1 with the data for inonophenol A, most of the data are similar, except for the C 10 , C 11 , C 12 , and -OCH 3 data of 1.

Results and Discussion
In 1, 1 H and 13 C NMR signals of an -OCH 3 [δ H 3.27 (3H, s); δ C 56.8)] were observed. However, in inonophenol A, there was no 1 H and 13 C NMR signal of -OCH 3 .
The above 1 H and 13 C NMR data (Table 1) of 2 indicated that there was also a phenylhexyl skeleton in 2, the same as in inonophenol A [12]. The molecular formula of 2 was C 12 H 14 O 3 (m/z 206), which has one less -H 2 O group (m/z 18) than inonophenol A (C 12 H 16 O 4 , m/z 224). Comparing the 1 H and 13 C NMR data of 2 with the data for inonophenol A, most of the data are similar, except for the C 8 , C 9 , C 10 , C 11 , and C 12 data of 2.
4 was obtained as a white amorphous powder, and its molecular formula was analyzed as C 18  The above 1 H and 13 C NMR data ( Table 2) of 4 indicate that there was a phenylhexyl skeleton in 4, the same as in inonophenol A [12]. The molecular formula of 4 was C 18 H 26 O 9 (m/z 386), which was one more -C 6 H 10 O 5 -group (m/z 162) than inonophenol A (C 12 H 16 O 4 , m/z 224). Comparing the 1 H and 13 C NMR data of 4 with data for inonophenol A, most of the data are similar, except for the C 10 , C 11 , C 12 , and -C 6 H 10 O 5 -data of 4. and 3.12-3.84; δ C 102.4 and 62.9-78.0 (C 1 -C 6 )] were observed. However, in inonophenol A, there were no 1 H and 13 C NMR signals of this.
The acid hydrolysis experiment on 4 afforded D-glucose, confirmed by TLC and a comparison of its NMR data with those of (5S)-1,7-bis-(3,4-dihydroxy-phenyl)-5-hydroxyheptan-3-one-5-O-β-D-glucopyranoside [13], and the relative configuration of the anomeric carbon to be β-configuration due to its large coupling constant. Based on the above evidence, the monosaccharide was determined to be β-D-glucopyranose.
The C 11 signal (δ C 72.4) in 4 was down-shifted by 7.3 chemical shift units compared to that in inonophenol A. Meanwhile, the C 10 and C 12 signals (δ C 51.6 and 20.3) in 4 were up-shifted by 1.1 and 3.2 chemical shift units, respectively, compared to those in inonophenol A. This evidence proved that 4 was a glycation product of inonophenol A by a β-D-glucopyranose moiety. The long-range correlations (Figure 2) of δ H 4.34 (1H, d, J = 7.2 Hz, 1 -H) with δ C 72.4 (C 11 ), and δ H 4.34 (1H, m, 11-H) with δ C 102.4 (C 1 ) in the HMBC spectrum of 4 further support this inference. Based on the above MS, 1 H, 13 C NMR, and HMBC spectra, it was confirmed that 4 is 3,4,11-trihydroxyphenylhex-9-one 11-O-β-D-glucopyranoside.
The absolute configuration of C 11 in 4 was determined to be R-configuration based on the contrast of the rotation value of the hydrolyzed aglycone ([α] 25 D − 4.56) of 4 with that of inonophenol A ([α] 20 D + 5.42) [12]. Therefore, 4 was identified as (R)-(−)-3,4,11trihydroxyphenylhex-9-one 11-O-β-D-glucopyranoside. 5 was obtained as a white amorphous powder, and its molecular formula was analyzed as C 18  The above 1 H and 13 C NMR data (Table 2) of 6 indicated that there was also a phenylhexyl glycoside skeleton in 6, the same as in 5. The molecular formula of 6 was C 18 H 28 O 8 (m/z 372), which was two -H-atoms (m/z 2) more than 5 (C 18 H 26 O 8 , m/z 370). Comparing the 1 H and 13 C NMR data for 6 with data for 5, most of the data are similar, except for the C 8 , C 9 , and C 10 data of 6.
In 5, the 13 C NMR signal [δ C 211.9] of a C=O was observed. However, in 6, there was no 13 C NMR signal of a C=O group. Instead, the 1 H and 13 C NMR signals [δ H 3.74 (1H, tt, J = 8.5, 4.3 Hz); δ C 70.1 (C 9 )] of one more methine group linking with oxygen were observed; meanwhile, the 13 C signal of C 9 (δ C 70.1) in 6 was up-shifted by 141.8 and the 13 C signals of C 8 and C 10 (δ C 40.8 and 45.5) were down-shifted by 5.4 and 6.0 chemical shift units, respectively, compared to those in 5. This evidence proves that 6 should be the product of the reduction of the carbonyl group in 5. The long-range correlations ( Figure 2) Molecules 2023, 28, 3928 7 of 12 of δ H 2.62 (2H, m, 7-H) and 4.10 (H, m, 11-H) with δ C 70.1 (C 9 ) in the HMBC spectrum of 6 further support this inference.
Based on the above MS, 1 H, 13 C NMR, and HMBC spectra, it was confirmed that 6 was phenylhex-4,9,11-triol 11-O-β-D-glucopyranoside. Due to technical limitations, the absolute configuration of 6 could not be determined.
7 was afforded as a white amorphous powder, and its molecular formula was analyzed as C 20  The 1 H and 13 C NMR data (Table 2) of 7 indicated that there was a phenylhexyl glycoside skeleton in 7, the same as in 6. The molecular formula of 7 was C 20 H 30 O 9 (m/z 414), which was one more -COCH 2 -group (m/z 42) than 6 (C 18 H 27 O 8 , m/z 372). Comparing the 1 H and 13 C NMR data of 7 with data for 6, most of the data are similar, except for the C 8 , C 9 , C 10 , and -COCH 3 data of 7.

The Antioxidant Activities of Compounds 1-7
Compounds 1-7 isolated from the title plant were tested for their antioxidant effects. The results of the antioxidant activity assays are listed in Table 3. Table 3. Results of the antioxidant activity assays of compounds 1-7 from the title plant (mean ± SD, n = 3).

Acid Hydrolysis of Compound 4
Dissolved 4 (4 mg) with 0.1 mL CH 3 OH was added to 4 mL of H 2 SO 4 aqueous solution (1 mol/L) and kept at 90 • C for 3 h. Adjusted the reaction solution to pH neutral with sodium hydroxide solution (1 mol/L), and then ethyl acetate eluate was added to extract the solution 3 times. An ethyl acetate phase and an aqueous phase were obtained. The aqueous phase permeated and condensed, and monosaccharides in the concentrated solution were confirmed by TLC (CHCl 3 -CH 3 OH-H 2 O = 3:2:0.1) and D-glucose (standard sample) [18]. The Rf value of D-glucose was 0.6.

Determination of Antioxidant Activity
DPPH and ABTS radical scavenging experiments were performed to measure the antioxidant activity of compounds 1-7 [19,20].

DPPH Radical Scavenging Assay
A 100 µL volume of DPPH anhydrous ethanol solution (120 µM) was added to 100 µL anhydrous ethanol sample solution (12.5, 25, 50, 100, 200, and 400 µM) in a 96-well plate. The mixture was allowed to react at room temperature for 30 min in the dark, and then the absorbance of the mixture at a wavelength of 517 nm was measured with a microplate reader. Three parallel experiments were conducted. DPPH radical scavenging activity was calculated using the following formula: DPPH scavenging activity was calculated by the following formula: DPPH scavenging activity (%) = (A control − A sample )/A control × 100%, where A control is the absorbance of the anhydrous ethanol control without samples, and A sample is the absorbance of sample. L-ascorbic acid was used as a positive control in the experiment.

ABTS Radical Scavenging Assay
A 100 µL volume of ABTS anhydrous ethanol solution (140 µM) was added to 100 µL anhydrous ethanol sample solution (12.5, 25, 50, 100, 200, and 400 µM) in a 96-well plate. The mixture was reacted at room temperature for 5 min in the dark, and then the absorbance of the mixture at a wavelength of 734 nm was measured with a microplate reader. Three parallel experiments were performed. The ABTS radical scavenging activity was calculated by the following formula: ABTS scavenging activity (%) = (A control − A sample )/A control × 100%, where A control is the absorbance of anhydrous ethanol control without samples, and A sample is the absorbance of the sample. L-ascorbic acid was used as a positive control in the experiment.

Statistical Analyses
The statistical analyses were performed using GraphPad Prism 8.0. Every sample was analyzed in triplicate. The IC 50 value of a compound (where half of DPPH and ABTS free radicals are cleared) was obtained by plotting the scavenging percentage of every sample of the compound against its concentration. The results are expressed as the mean ± standard deviation (SD). The difference in the means between compound and positive control was analyzed by one-way analysis of variance (ANOVA) using SPSS 25.0, to judge whether there was a statistically significant difference between the groups (p < 0.05).

Conclusions
To date, only three similar components (inositol A, inositol B, and hispolon) similar to the skeleton of the compounds reported in this paper have been reported to have been isolated from microorganisms [12,21]. However, the original literature defines them as being of the phenyl-substituted hexane type. This paper reports these compounds from plants for the first time, and based on the skeleton naming rule of natural products, the skeleton type was denoted as phenylhexanoid. This name is more in line with its biosynthetic pathway.
Although compounds 1-7 showed some antioxidant activities, further research is needed to see if they have an effect when used for the treatment of hepatitis, cholecystitis, and digestive diseases.